Terminally differentiated epidermal keratinocytes form the body's outer protective barrier that undergoes constant renewal as cornified squames slough off the exterior surface and are replaced by material migrating outwards from the inner living layers of the epidermis. Cornified squames are composite biomaterials with two components: a matrix of keratin filaments encased within the cornified cell envelope (CE). The CE is a multi-component 10nm-thick layer of insoluble protein deposited on the inner surface of the plasma membrane of the cells during terminal differentiation. In the epidermis, a 5nm-thick layer of ceramide lipids (lipid envelope) is attached to the exterior surface. The insolubility of the protein envelope is due in large part to the cross-linking of several structural proteins by transglutaminases. Intermediate filaments (IF) are ubiquitous constituents of the cytoskeletons of eukaryotic cells. They consist of five different types, of which the most numerous and complex are the type I and type II keratins widely expressed in epithelia. We are interested also in the related IF of other cell types and in the interactions between keratin IF and the CE. The long-term goal of this project, previously pursued in both the Laboratory of Skin Biology (P. M. Steinert, P.I.) and the LSBR is to elucidate the production, assembly, structure and biomechanical properties of these cells and their components in the context of both normal and diseased skin. This project is now largely in abeyance, but we did succeed in FY 10 in completing and submitting for publication a paper reporting the following results. In normal skin, some 75% of the protein mass of the CE is contributed by a single protein called loricrin. Despite the functional importance that this number would suggest, knocking out the loricrin gene in mice results in only a mild transient phenotype and the loricrin knockout CE maintains a normal thickness (15nm). However, an examination of the loricrin KO CE amino acid composition revealed changes in the amino acid content, suggesting that other proteins were incorporated into the CE to compensate for its loss. Recently our collaborator has determined that several members of the late cornified envelope (lce) protein family are significantly upregulated at the mRNA and protein level in the KO mouse, and these proteins are incorporated into the CE to functionally compensate for the loss of loricrin. We have generated immuno-gold EM data with anti-lce antibodies to confirm and extend these observations. This paper is currently in revision.